Compositions containing cobalt hyaluronic acid complex

ABSTRACT

Complexes of deprotonated hyaluronic acid with 3d metal ions of the 4th period of the Periodic Table and compositions containing these complexes as active ingredients or carriers. A process for the preparation of the complexes and compositions (pharmaceutical and cosmetic compositions) containing these complexes as active ingredients are disclosed in which zinc or cobalt (II) hyaluronate is preferably used as active ingredient.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-in-Part of copending application Ser.No. 07/928,154 filed 10 Aug. 1992 as continuation of then copendingapplication Ser. No. 07/602,326 filed 21 Nov. 1990 as a National Phaseapplication of PCT/HU90/00013 filed 20 Feb. 1990 and based uponHungarian national application Ser. No. 891/89 of 24 Feb. 1989 under theInternational Convention.

FIELD OF THE INVENTION

This invention relates to novel hyaluronic acid associates (complexes)of 3d metal ions of the 4th period of the Periodic Table, with astoichiometric composition, prepared through the interaction ofequivalent amounts of an alkali metal hyaluronate, alkaline earth metalhyaluronate, silver hyaluronate or a quaternary ammonium hyaluronate and3d metal ions in aqueous solution, and to pharmaceutical compositionscontaining these associates (complexes) as active ingredients.

The invention further relates to a process for the preparation of thesenovel associates (complexes) and compositions containing theseassociates (complexes) as active ingredients.

According to a particularly preferred embodiment of the process of thepresent invention, the aqueous solutions containing the novel hyaluronicacid associates (complexes) of 3d metal ions of the 4th period of thePeriodic Table, with a stoichiometric composition, prepared through theinteraction of equivalent amounts of sodium hyaluronate, and 3d metalions in aqueous solution, are prepared directly from an aqueous solutionof sodium hyaluronate.

The novel associates according to the present invention mainly involvezinc and cobalt hyaluronate. The compositions containing these latterassociates may be pharmaceutical (therapeutical) or cosmetic andoptionally other compositions. The compositions containing the novelassociates according to the invention are therapeutically effective fore.g.: the acceleration of epithelization of epithelium-deficient bodysurfaces; healing of crural ulcers, decubitus (bed-ulcers), primarilynot healing wounds, burns, radiation- or heat-induced wounds, vulgaracne and conglobated acnes, although they can be used in other areas,too.

BACKGROUND OF THE INVENTION

Hyaluronic acid is a macromolecule known for more than 50 years andwhich has first been described by Meyer et al. [J. Biol. Chem. 107, 629(1954); J. Biol. Chem. 114, 689 (1936)]. The structure determination wasperformed by Weissman et al. [J. Am. Chem. Soc. 76, 1753 (1954)].Hyaluronic acid is a highly viscous native glucosaminoglycan containingalternating β₁₋₃ glucouronic acid and β₁₋₄ glucosamine moieties; itsmolecular weight is between 50000 and several (8 to 13) millions. Therecovery of hyaluronic acid is an old task. The separation and use of anextra pure hyaluronic acid are described e.g. in the U.S. Pat. Nos.4,141,973 and 4,303,676 and in the European Patent No. 0 144 019. Untilrecently hyaluronic acid has been employed as the sodium salt e.g. intherapy, mainly in opthalmology, surgery and cosmetics. The salts ofhyaluronic acid formed with alkali, alkaline earth, magnesium, aluminum,ammonium or substituted ammonium ions may serve as carriers forpromoting the absorption of drugs (see the Belgian Patent SpecificationNo. 904,547). Heavy metal salts of hyaluronic acid (wherein "heavymetals" mean the elements of the 5th, 6th and 7th periods of thePeriodic Table as well as the lanthanides and actinides) and withinthese the silver salt are utilized as fungicidal agents whereas the goldsalt is employed for the treatment of arthritis (see the patentspecification WO 87/05517).

It has been proven by various structure-elucidating methods that thesecondary structure, i.e. the conformation of hyaluronic acid is changedby binding metal ions [W. T. Winter and A. Struther: J. Mol. Biol. 517,761 (1977); J. K. Sheehan and E. D. T. Atkins: Int. J. Biol. Macromol.5, 215 (183); and N. Figueroa and B. Chakrabarti: Biopolymers 17, 2415(1978)]. Significantly varying effects on the molecular structure can beexerted even by metal ions of similar character as shown by comparativeX-ray study of potassium and sodium hyaluronate [A. K. Mitra et al.: J.Macromol., Sci. Phys. 824, 1 and 21 (1985)]. This is all the more validfor compounds of hyaluronic acid formed with metal ions of various sortsbearing various charges.

No reference relating to hyaluronic acid associates (complexes) of 3dmetal ions of the 4th period of the Periodic Table, with astoichiometric composition, prepared through the interaction ofequivalent amounts of an alkali metal hyaluronate, alkaline earth metalhyaluronate, silver hyaluronate or a quaternary ammonium hyaluronate and3d metal ions in aqueous solution, can be found in the literature.Actually, according to gel filtration chromatography examinations,hyaluronic acid, in contrast with heparin, is unable to bind zinc ions[(R. F. Parish and W. R. Fair: Biochem. J. 193, 407 to 410 (1981)].

In spite of the fact that, according to the literature, hyaluronic acid(or its sodium salt) is unable to bind zinc ions, we undertook toinvestigate the coordination chemistry of the interaction betweenhyaluronic acid and 3d metal ions of the 4th period of the PeriodicTable and among these, chiefly, zinc and cobalt ions. Since hyaluronicacid is nearly exclusively commercialized as its sodium salt thus beingthe basic substance of all systems containing hyaluronate, ourinvestigations were begun on the interaction of sodium ions andhyaluronate. For this purpose the free sodium ion activity of aqueoussodium hyaluronate solutions was measured by using a sodium selectiveglass electrode. It was unambiguously found from these measurements thatnot more than 60% of sodium ions introduced as equivalent together withthe carboxylate groups of hyaluronate are present as free ions in theaqueous solutions whereas the remainder of 40% is in a form bound to thehyaluronate.

According to our measurements, by increasing the sodium ionconcentration the amount of the sodium ions bound can be raised to50-55% calculated for all available carboxylate groups. Thus, it hasbeen verified that, as contrasted with common properties of salts,sodium hyaluronate is not completely dissociated in aqueous solution.

DESCRIPTION OF THE INVENTION

In the next step of our investigations an aqueous solution of sodiumhyaluronate was titrated with zinc chloride solution by using a sodiumion-selective electrode as mentioned above for following the change inthe activity of free sodium ions in the system. A characteristic curvereflecting the process is shown in FIG. 1. It is perceivable that sodiumions originally bound to hyaluronate are liberated on the effect of zincions. Based on the results of these measurements the total sodium ionconcentration is liberated by an equivalent amount of zinc, a factunequivocally proving that zinc ions are more strongly bound tohyaluronate than are sodium ions. Thus, the earlier statement thathyaluronic acid would be unable to bind zinc ions [R. F. Parrish and W.R. Fair: Biochem. J. 193, 407 (1981)] has experimentally been refuted.

Thereby, knowledge previously held by workers skilled in the art wasdisproved.

From our investigations discussed above it became clear that, throughthe interaction of equivalent amounts of sodium hyaluronate and zincions (zinc chloride) in aqueous solution a zinc hyaluronate associatewith a stoichiometric composition is formed. After an appropriateisotonization the solution obtained can directly be used fortherapeutical purposes and the zinc compound need not be prepared insolid state in a separate process.

Nevertheless, the complex was prepared in a solid state forcharacterization and the direct environment of the zinc ion wasdetermined by using the "Extended X-ray Absorption Fine Structure"(EXAFS) method. It has been found that zinc is surrounded by four oxygenatoms in the first coordination sphere. The length of the Zn--O bonddistances is 199 pm whereas two carbon atoms are present in a longerdistance of 241 pm from the zinc atom.

According to our examinations zinc hyaluronate significantly differsfrom the analogous copper complex which latter contains four equatorialand two axial Cu--O bonds with the values of 194 and 234 pm,respectively. The distance between the copper atom and the new twocarbon atoms is 258 pm. The structure of the cobalt (II) complex issimilar to the zinc complex but not to the copper complex; specificallythe Co--O bond distance is 197 pm±1.0 and the Co--C is 239 pm ±1.5 pm.

The present invention relates to hyaluronic acid associates (complexes)of 3d metal ions of the 4th period of the Periodic Table, with astoichiometric composition, prepared through the interaction ofequivalent amounts of alkali metal hyaluronate, alkaline earth metalhyaluronate, silver hyaluronate or a quaternary ammonium hyaluronate and3d metal ions in aqueous solution.

The invention further relates to a pharmaceutical composition containingas active ingredient hyaluronic acid associates (complexes) of 3d metalions of the 4th period of the Periodic Table, with a stoichiometriccomposition, prepared through the interaction of equivalent amounts ofan alkali metal hyaluronate, alkaline earth metal hyaluronate, silverhyaluronate or a quaternary ammonium hyaluronate and 3d metal ions inaqueous solution, optionally in admixture with a carrier and/or otheractive ingredients and/or additives.

According to another aspect of the invention, there is provided aprocess for the preparation of the novel associates (complexes) of theinvention, with a stoichiometric composition, which comprises:

a) adding an aqueous solution containing the equivalent amount of asalt, preferably the chloride of one of the 3d metal ions of the 4thperiod of the Periodic Table, to an equivalent amount of an aqueoussolution of sodium hyaluronate or to an equivalent amount of anothersalt (alkali or alkaline earth metal salt, optionally silver salt) ofhyaluronate; or

b) dissolving an associate formed from hyaluronic acid with a quaternaryammonium salt in an aqueous suspension in a solvent couple containingthe aqueous solution of an equivalent amount of a 3d metal ion of the4th period of the Periodic Table and a solvent which is precipitatingthe hyaluronic acid associates (complexes) of 3d metal ions of the 4thperiod of the Periodic Table, as stoichiometric compositions, by analkanol or alkanone in a known manner, or

separating the precipitate from the solution and then, if desired

drying it under mild conditions.

This process serves for the preparation of aqueous solutions containingas active ingredient a stoichiometric composition which is a zinc orcobalt (II) hyaluronate associate (complex) or a similar associate of a3d metal ion of the 4th period of the Periodic Table, respectively.These solutions were in each case prepared by the direct reaction of themetal ion with the hyaluronate component. This method of preparationmade unnecessary to previously separate the active ingredients mentionedabove in a solid state. In the solution prepared by using the process ofthe invention the amount of free (metal-unbound) hyaluronate isnegligible even in the presence of an equivalent amount of zinc. In thepresence of an excess of zinc ions the formation of the zinc hyaluronateassociate (complex) becomes quantitative.

In the course of preparation of the metal associates as discussed abovethe pH remains at a value of about 4.5 to 6.5. In the case of a 0.2% byweight/volume (wt./vol.) hyaluronate solution the pH reaches a value of5.4 whereas in the case of 0.5% by wt./vol. the pH value is 5. Whennecessary, the pH of the latter system can be adjusted to a value of 5.5to 5.6 by adding a few drops of isotonic sodium acetate solution.

Solutions of two sorts containing zinc hyaluronate as active ingredienthave been prepared by using the process discussed above.

1. Zinc hyaluronate solution made isotonic by an excess of zincchloride;

Taking into consideration that free zinc chloride alone may alsopreferably be used in the dermatology, the osmotic pressure of the zinchyaluronate solution was adjusted to the isotonic value by using anexcess of zinc chloride. The solution thus obtained did not contain anyfree (zinc-unbound) hyaluronate at all but an excess of zinc chloridewas present in the system together with zinc hyaluronate.

2. Zinc hyaluronate solution made isotonic by a monosaccharide or asugar alcohol:

For a therapeutic use wherein the presence of hyaluronate-unbound zincions is not indicated, the stoichiometric solution containing zinc ionsin an amount equivalent to the hyaluronate was made isotonic by using apolyalcohol (sugar alcohol, preferably sorbitol) or a mono-ordisaccharide (preferably glucose). The free zinc ion and freehyaluronate content of these latter systems did not reach 5% of thetotal zinc or total hyaluronate content, respectively.

In the course of utilizing the associates according to the inventionion-free compositions may eventually be required. Namely, the associatesprepared according to the above process of the invention usually containsodium chloride or another salt formed from the starting hyaluronatecation and the anion of the 3d metal salt.

Two different process variants can be used for the preparation of asalt-free hyaluronic acid associate formed with a 3d metal ion. Theseare as follows.

a) A solution of a quaternary ammonium salt is portionwise added to thesolution of a known hyaluronate, preferably sodium hyaluronate. After asatisfying purification, the novel quaternary ammonium hyaluronateassociate precipitated is dissolved under vigorous stirring in a solventcouple consisting of an aqueous solution of a 3d metal ion of the 4thperiod of the Periodic Table and a solvent which separate, then thehyaluronate associate is precipitated by adding an alkanol or alkanoneto the aqueous phase, the precipitate is separated and washed; or

b) after adding 2.0 to 3 volumes of a C₁₋₃ alkanol or C₃₋₄ alkanoneunder stirring to a zinc hyaluronate solution, suitably to a notisotonized solution containing zinc chloride in an amount equivalent tothe hyaluronate, the zinc hyaluronate precipitated is filtered andwashed with the alkanol or alkanone, respectively used for theprecipitation. When necessary, the zinc hyaluronate is dissolved inion-free water and the precipitation is repeated.

When a solid ion-free zinc hyaluronate is needed, the precipitate isdried under reduced pressure under mild conditions. In the case of ademand on an ion-free zinc hyaluronate solution it is preferable todissolve the zinc hyaluronate made free from the solvent. According toany of both process variants an ion-free solid or dissolved product isobtained with an optional purity depending on the quality of thestarting zinc hyaluronate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a titration curve showing the titration of an aqueous sodiumhyaluronate solution with zinc

FIG. 2 is a series of two graphs showing the results of the clinicaltreatment of patients suffering from crural ulcers using zinchyaluronate according to the curve marked with a cross and using sodiumhyaluronate using the curve marked with a square. The x-axis shows theduration of treatment and the y-axis shows the severity of the disease.

FIG. 3 is a series of bar graphs showing the severity of crural ulcerstreated with sodium hyaluronate and zinc hyaluronate, respectively, overperiods of 1, 2, 3 and 4 weeks.

The results of the clinical-pharmacological investigation on acomposition (Example 13) containing as active ingredient the zinchyaluronate according to our invention are shown on a crural ulcertreatment used for the acceleration of epithelization ofepithelium-deficient surfaces. A composition containing sodiumhyaluronate was used as control.

This examination was carried out on 12 or 14 ulcers, respectively of 8or 12 patients suffering from crural ulcer. The distribution of thepatients of both groups according to sex and age as well as to thenature of the disease was as follows.

    ______________________________________                                                                                 Character                                    No. of              Aver- No. of of the                               Active  Pa-     Wo-         age   Ulcers Ulcer.sup.x                          Ingredient                                                                            tients  man    Man  Age   Treated                                                                              A   V   M                            ______________________________________                                        Zinc    12      10     2    63.9  14     2   9   1                            hyaluronate                                                                   Sodium   8       6     2    65.7  12     --  7   1                            hyaluronate                                                                   ______________________________________                                         .sup.x A = arterial; V = venous; M = Mixed                               

The treatments were performed in such a way that before beginning thetreatment a purifying therapy was carried out according to the actualclinical state of the ulcer. The treatment with zinc or sodiumhyaluronate, respectively, was commenced on ulcers nearly purified or incases where a significant diminution of the sorbes was observed. Thetreatment was carried out daily once in such a way that the compositionwas dropped onto the surface of the purified ulcer in am amount wettingthe surface of the wound with a thin layer.

The composition was used for 4 weeks. At the beginning of the treatmentand then once in a week the data sheet was filled out and the ulcers ofthe patients were documented by photographs. A discharge sample wastaken for bacteriological examination.

The characteristics as well as the severity of the epithelial lesionswere marked with the following symbols and scores.

    ______________________________________                                        Characteristics        Severity                                               ______________________________________                                        Area (a)                                                                      0                      0                                                      Below 10 cm.sup.2      1                                                      Between 10 cm.sup.2 and 25 cm.sup.2                                                                  2                                                      Above 25 cm.sup.2      3                                                      Infectedness (b)                                                              Clinically pure        0                                                      Coated in 50%          1                                                      Coated in 100%         2                                                      Necrosis (c)                                                                  (only in the case of an arterial ulcer)                                       negative               0                                                      Below 10%              1                                                      Between 10% and 15%    2                                                      100%                   3                                                      No necrosis            4                                                      ______________________________________                                    

EVALUATION

For evaluation the values of the separate characteristics weredetermined and the general severity score was calculated by using thefollowing formula:

    s=a×b×c

The results of the clinical pharmacological investigations areillustrated in FIG. 2. The results of the treatment with zinchyaluronate is shown on the curve marked with a cross whereas that ofthe treatment with sodium hyaluronate is illustrated on the curvedenoted with a square as a function of the number of weeks involving thetreatment. The score value plotted on the ordinate represents thegeneral severity index calculated by using the above formula.

For a more correct comparison of zinc hyaluronate to sodium hyaluronateused as control the relative correct values related to the startingscore values as 100% are illustrated in FIG. 3.

The change in the relative correct values was statistically evaluated asa function of number (1 to 4) of weeks. On the zinc and sodiumhyaluronate treatment, the number of ulcers decreased below a relativescore value of 90%, 80%, 70% and 60%, respectively after 1, 2, 3 and 4weeks was investigated. The results are summarized in Table 1.

                                      TABLE 1                                     __________________________________________________________________________             1st week                                                                              2nd week                                                                              3rd week                                                                              4th week                                              Distribution of the relative score value                             Active ingredient                                                                      90%     80%     70%     60%                                          of the composition                                                                     below                                                                             above                                                                             below                                                                             above                                                                             below                                                                             above                                                                             below                                                                             above                                    __________________________________________________________________________    Zinc     12  2   11  3   11  3   11  3                                        hyaluronate                                                                   Sodium   4   8   7   5   6   6   3   9                                        hyaluronate                                                                   __________________________________________________________________________

It can be stated from Table 1 that the treatment with zinc hyaluronatewas in every week advantageous in comparison to the results obtainedwith sodium hyaluronate used as control.

The statistical analysis of the response obtained for the hypothesis inquestion proved that the advantage of the zinc hyaluronate compositionwas highly significant (p. 99%) in comparison to sodium hyaluronate.

In a further statistical working-up, a more detailed distribution of therelative score values was investigated as a function of the time oftreatment. The results obtained are summarized in Table 2.

                  TABLE 2                                                         ______________________________________                                                  Number and score value of the ulcers                                Active ingredient                                                                         above   between   between below                                   of the composition                                                                        90%     90 and 70%                                                                              70 and 50%                                                                            50%                                     ______________________________________                                        1st week                                                                      Zinc hyaluronate                                                                          2       7         5       0                                       Sodium hyaluronate                                                                        8       3         0       1                                       2nd week                                                                      Zinc hyaluronate                                                                          0       6         7       1                                       Sodium hyaluronate                                                                        4       3         5       0                                       3rd week                                                                      Zinc hyaluronate                                                                          0       1         8       2                                       Sodium hyaluronate                                                                        2       5         5       1                                       4th week                                                                      Zinc hyaluronate                                                                          2       1         7       3                                       Sodium hyaluronate                                                                        1       5         3       3                                       ______________________________________                                    

The data of Table 2 similarly support the advantage of zinc hyaluronate.The more detailed statistical examinations show the significance todecrease depending on the time of treatment.

Summing up: on an evaluation of the clinical-pharmacologicalinvestigations the higher efficiency of zinc hyaluronate could be proveneven at a low number of ulcers; this advantage could particularly besupported in the starting period of the treatment.

Microbiological examination of solutions were carried out. The solutionscontained either zinc hyaluronate (Zn--HY) prepared according to thepresent invention, sodium hyaluronate (Na--HY) obtained as HEALON™(Pharmacia), or Na--HY containing zinc ions prepared according toExample 5 of Czech Inventor's Certificate 264,719.

The following example shows preparation of a 0.1% aqueous solution ofZn--HY according to the present invention:

EXAMPLE A

Preparation of a zinc hyaluronate solution 40.18 mg of sodiumhyaluronate are dissolved in 20.0 ml of twice distilled water. Thus, thestarting concentration of hyaluronic acid is 2.009 mg/ml, the equivalentconcentration of the solution is 4.241×10³ mol/liter (Na⁺ or hyaluronicacid dimer unit). In the course of the measurement, a zinc chloridesolution of 0.05154 mol/liter concentration is added to the reactionmixture through a microburet. The solution is first added in littleportions (0.05 ml) and then in larger portions (0.1 to 0.2 ml). Thepotential change in the solution is measured by using a precisionpotentiometer with digital display and sodium-selective glass andsilver/silver chloride electrodes. The titration is continued until thepotential measured is not further changed by adding an additionalportion of the titrating solution. (The measuring system was calibratedunder conditions analogous to the practical measurement). A 0.1% aqueoussolution of zinc hyaluronate was obtained.

The selectivity of the sodium ion-selective electrode was observed alsoin the presence of Zn²⁺ ions in order to control that the potentialchange in the practical measurement was caused by the liberated Na⁺ ionsand not the Zn²⁺⁰ ions introduced to the solution. A 2.00×10⁻³ M sodiumchloride solution was titrated by using the zinc chloride titratingsolution under conditions similar to the above conditions. On increasingthe concentration of Zn²⁺ from 0 up to 4×10⁻³ mol/liter a potentialincrease of about 2 mV was observed whereas the practical measurementshowed a change of about 20 mV under similar conditions. Thus, theevaluation had no obstacle. In the course of measurement the increase inthe sodium ion

Preparation of a zinc chloride solution

since a solution containing zinc chloride in an accurate concentrationcannot be prepared by direct weighing-in, first a solution with thenearly desired concentration is prepared. On preparing this solution noacid should be used thus it may occur that the zinc chloride weighed inwill not completely be dissolved. After sedimentation of the insolubleresidue (about 30 minutes) the volumetric flask is filled up to the markand the solution is filtered through a filter paper.

The accurate concentration of the filtrate is determined bycomplexometric titration by using buffer of pH 10 and eryochrom black-Tindicator. The zinc chloride solution with an accurate concentration of0.100 mol/liter is prepared by the precise dilution of this solution.

The characteristics of sodium hyaluronate used for the preparation ofsolution are as follows:

    ______________________________________                                        Molecular weight     1850000 daltons                                          Protein content      0.07% by wt.                                             UV absorption                                                                 A.sub.257.sup.1 %    0.133                                                    A.sub.280.sup.1 %    0.075                                                    Viscosity [η] .sub.25° C..sup.c→0 %                                              13.7 dl/g                                                HA.sup.x             98.12% by wt.                                            ______________________________________                                         .sup.x HA = hyaluronic acid (as abbreviated herein).                     

The following example shows preparation of a 0.1% aqueous solution ofsodium hyaluronate containing zinc ions disclosed in CzechoslovakianInventor's Certificate 264,719:

EXAMPLE B (Example 5 of Czech Inventor's Certificate)

A 0.1% aqueous solution of Na--HY containing zinc ions is prepared bydissolving 10 mg of Na--HY and 0.001 mg of zn(SO₄). 7H₂ O in 10 ml ofapyrogenic physiological solution.

Method: One ml of the different 0.1% aqueous starting samples wasinoculated with 1 ml of the test organisms described herein below andthe changing of the number of germs was taken against time.

The number of germs was determined in a point of time by plating method.

Medium applied: Soya-casein agar (Caso-agar [Merck]).

Microorganisms applied: Staphylococcus aureus Salmonella sp.

Starting number of germs: Σ10⁶ /ml

Number of parallel experiments: 3

The solutions applied:

1--aqueous solution of Zn--HY of 0.1% by weight.

2--aqueous solution of Na--HY of 0.1% by weight.

3--aqueous solution of Na--HY containing Zn ions (0.1% by weight)(prepared according to the process described in the Czecho-SlovakianInventor's Certificate No. 264 719).

The results are shown in Table 1.

As it is shown, in the case of Zn--HY no germs were observed after 48hours but in the case of Na--HY and Na--HY containing Zn ions the numberof germs began to increase after 24 hours.

                                      TABLE 3                                     __________________________________________________________________________    Staphylococcus aureus                                                                              Salmonella sp.                                           Time 1    2    3     1    2     3                                             __________________________________________________________________________    0 h  4.2 × 10.sup.6                                                               1.3 × 10.sup.6                                                               1.4 × 10.sup.6                                                                2.0 × 10.sup.6                                                               8.0 × 10.sup.6                                                                4.2 × 10.sup.6                          2 h  3.4 × 10.sup.6                                                               1.4 × 10.sup.6                                                               1.3 × 10.sup.6                                                                1.4 × 10.sup.6                                                               7.0 × 10.sup.6                                                                4.4 × 10.sup.6                          4 h  3.1 × 10.sup.6                                                               1.2 × 10.sup.6                                                               1.3 × 10.sup.6                                                                1.2 × 10.sup.6                                                               6.8 × 10.sup.6                                                                4.2 × 10.sup.6                          6 h  2.4 × 10.sup.6                                                               1.6 × 10.sup.6                                                               1.2 × 10.sup.6                                                                1.0 × 10.sup.6                                                               6.5 × 10.sup.6                                                                3.9 × 10.sup.6                          1 day                                                                              2.6 × 10.sup.6                                                               4.1 × 10.sup.6                                                               2.1 × 10.sup.6                                                                1.1 × 10.sup.6                                                               8.1 × 10.sup.6                                                                6.1 × 10.sup.6                          2 day                                                                              0    6.2 ×  10.sup.6                                                              4.5 × 10.sup.6                                                                0    1.2 × 10.sup.6                                                                1.3 × 10.sup.6                          3 day                                                                              0    8.4 × 10.sup.6                                                               8.4 × 10.sup.6                                                                0    1.8 × 10.sup.6                                                                1.7 × 10.sup.6                          4 day                                                                              0               0                                                        7 day                                                                              0               0                                                        __________________________________________________________________________

EXAMPLE C

The test for the antibacterial effect of Co(II)HY has also been carriedout and the results are given in the following table. The test wascarried out in the same was as in Example A except that E coli wasapplied as the second microorganism instead of Salmonella sp.

                  TABLE 4                                                         ______________________________________                                        Staphylococcus aureus    E coli.                                              Time    1        2           1      2                                         ______________________________________                                        0 h       3 × 10.sup.6                                                                   1.9 × 10.sup.6                                                                      4.5 × 10.sup.6                                                                 5.8 × 10.sup.6                      4 h     2.1 × 10.sup.6                                                                   7.5 × 10.sup.5                                                                      4.6 × 10.sup.6                                                                 4.5 × 10.sup.6                      24 h    1.3 × 10.sup.6                                                                   3.5 × 10.sup.6                                                                      1.8 × 10.sup.6                                                                 1.0 × 10.sup.6                      48 h    5.8 × 10.sup.6                                                                   1.0 × 10.sup.6                                                                      5.5 × 10.sup.6                                                                 9.0 × 10.sup.2                      72 h    2.8 × 10.sup.6                                                                   <10         5.5 × 10.sup.6                                                                 1.0 × 10.sup.2                      6 days  1.5 × 10.sup.6                                                                   <10         1.3 × 10.sup.6                                                                 <10                                       7 days  1.6 × 10.sup.6                                                                   <10         2.5 × 10.sup.6                                                                 <10                                       ______________________________________                                         1: Aqueous 0.1% by weight solution of NaHY.                                   2. Aqueous 0.1% by weight solution of CoHY.                              

Note that in Examples A, B and C, the molecular weight of the hyaluronicacid in each case was 1,850,000 daltons. In our opinion, hyaluronic acidwill be expected to function equivalently in the present inventionirrespective of whether its molecular weight is as low as 50,000 or ashigh as 8 to 13 million daltons.

Specific Examples

The invention is illustrated in more detail by the following nonlimiting Examples.

The protein content of hyaluronate (HA) was determined by using themethod of O. H. Lowry [J. Biol. Chem. 193 (1951)]; the viscosity ofhyaluronate was measured in an Ostwald's viscometer in a physiologicalsaline solution at 25° C. The value of the intrinsic viscosityextrapolated to "0" concentration, i.e. [η] ##STR1## is given below. TheHA content was determined by using Bitter's method [Anal. Biochem. 4,330 (1962)].

EXAMPLE 1

Preparation of a zinc hyaluronate solution

40.18 mg of sodium hyaluronate are dissolved in 20.0 ml of twicedistilled water. Thus, the starting concentration of hyaluronic acid is2,009 mg/ml, the equivalent concentration of the solution is 4,241×10⁻³mol. liter (Na⁺ or hyaluronic acid dimer unit). In the course of themeasurement, a zinc chloride solution of 0.05154 mol/liter concentrationis added to the reaction mixture through a microburet. The solution isfirst added in little portions (0.05 ml) and then in larger portions(0.1 to 0.2 ml. The potential change in the solution is measured byusing a precision potentiometer with digital display and sodiumion-selective glass and silver/silver chloride electrodes. The titrationis continued until the potential measured is not further changed byadding an additional portion of the titrating solution. (The measuringsystem was calibrated under conditions analogous to the practicalmeasurement).

The selectivity of the sodium ion-selective electrode was observed alsoin the presence of Zn²⁺ ions in order to control that the potentialchange in the practical measurement was caused by the liberated Na⁺ ionsand not the Zn²⁺ ions introduced to the solution. A 2.00×10⁻³ M sodiumchloride solution was titrated by using the zinc chloride titratingsolution under conditions similar to the above conditions. On increasingthe concentration of Zn²⁺ from 0 up to 4×10⁻³ mol/liter a potentialincrease of about 2 mV was observed whereas the practical measurementshowed a change of about 20 mV under similar conditions. Thus, theevaluation had no obstacle. In the course of measurement the increase inthe sodium ion activity calculated from the measurement data verifiedthe quantitative formation of the zinc associate.

Preparation of a zinc chloride solution

Since a solution containing zinc chloride in an accurate concentrationcannot be prepared by direct weighing in, first a solution with thenearly desired concentration is prepared. On preparing this solution noacid should be used thus it may occur that the zinc chloride weighted inwill not completely be dissolved. After sedimentation of the insolubleresidue (about 30 minutes) the volumetric flask is filled up to the markand the solution is filtered through a filter paper.

The accurate concentration of the filtrate is determined bycomplexometric titration by using buffer 10 and eryochrom black-Tindicator. The zinc chloride solution with an accurate concentration of0.100 mol/liter is prepared by the precise dilution of this solution.

The characteristics of sodium hyaluronate used for the preparation ofsolution are as follows:

    ______________________________________                                        Molecular weight     1850000 daltons                                          Protein content      0.07% by wt.                                             UV absorption                                                                 A.sub.257.sup.1 %    0.133                                                    A.sub.280.sup.1 %    0.075                                                    Viscosity [η] .sub.25° C..sup.c→0 %                                              13.7 dl/g                                                HA.sup.x             98.12% by wt.                                            ______________________________________                                         .sup.x HA = hyaluronic acid (as abbreviated herein).                     

EXAMPLE 2

Preparation of a solution for dermatologic and cosmetic use

12.5 ml of a zinc chloride solution of 0.100 mol/liter concentrationprepared with ion-free water are added to 0.50 g of sodium hyaluronateweighted in a 100 ml volumetric flask. (Another concentration of zincchloride may also be used but the amount of zinc chloride should be thesame.) Sodium hyaluronate is allowed to swell (for 12 hours) in thesolution filled up to the mark with ion-free

The characteristics of sodium hyaluronate used for preparing the abovesolution are as follows:

    ______________________________________                                        Viscosity [η] .sub.25° C..sup.c→0 %                                               16.5 dl/g                                               Protein content       0.8% by wt.                                             ______________________________________                                    

EXAMPLE 3

Preparation of a zinc hyaluronate solution for use in injectablesolutions.

The operations described in this Example are carried out under sterileconditions.

5.0 ml of a zinc chloride solution of 0.100 mol/liter concentrationprepared with twice distilled water (water for injection use,pyrogen-free, sterile) are added to 0.20 g of sodium hyaluronate (ofpure powder quality) weighted in a 100 ml volumetric flask, then thevolume is filled up to 50 ml with twice distilled water. Sodiumhyaluronate is allowed to swell overnight, then dissolved by shaking andthe solution filled up to the mark with twice distilled water. Thesolution obtained is filtered through a membrane filter (0.45/μ poresize) to give a zinc hyaluronate solution of 0.2% by wt./vol.

The characteristics of the sodium hyaluronate used for preparing theabove solution are as follows:

    ______________________________________                                        Quality             pure, pyrogen-free                                        sterile powder                                                                Molecular Weight    1850000                                                   Protein content     0.07% by wt.                                              UV absorption                                                                 A.sub.257.sup.1 %   0.133                                                     A.sub.280.sup.1 %   0.075                                                     HA content          98.12% by wt.                                             Viscosity [η] .sub.25° C..sup.c→0 %                                             13.7 dl/g                                                 ______________________________________                                    

EXAMPLE 4

Preparation of an ion-free zinc hyaluronate solution

600 ml of ethanol of analytical grade are added under stirring to 200 mlof 0.50 % by wt./vol. zinc hyaluronate solution obtained according toExample 2, the precipitated zinc hyaluronate is filtered on a glassfilter, washed twice with 50 of ethanol each of the same quality andthen dried under reduced pressure. Thus, 0.88 g of zinc hyaluronate isobtained which is used for preparing a 0.50% by wt./vol. zinchyaluronate solution in the way described in Example 2. The zinchyaluronate solution obtained does not contain any sodium chloridearising from the reaction between sodium hyaluronate and zinc chloride;thus, it is practically ion-free.

EXAMPLE 5

Preparation of ion-free zinc hyaluronate or its solution fortherapeutical use

The operation described in this Example is carried out under sterileconditions.

1500 ml of ethanol (purest quality) are portionwise added to 500 ml ofzinc hyaluronate solution prepared according to Example 3 understirring. After the addition the system is stirred for 30 minutes, thezinc hyaluronate precipitate is filtered on a glass filter, washed 3times with 100 ml of ethanol (purest quality) each and dried underreduced pressure under mild and sterile conditions.

EXAMPLE 6

Preparation of ion-free zinc hyaluronate

200 ml of 10% by wt. solution of Hyamine® 1622 (puriss)(benzyldimethyl{-2-[2-p-(1,1,3,3-tetramethylbutyl)phenoxy]ethoxy}ethyl-ammonium chloride) are added under stirring to thesolution containing 1 g of sodium hyaluronate in 400 ml of twicedistilled water. The precipitate i.e. the hyaluronic acid quaternaryammonium associate formed is separated by centrifuging, washed twicewith 100 ml of twice distilled water each and again centrifuged. Thewashed precipitate is dissolved in a solvent couple consisting of 400 mlof 2% by wt./vol. zinc chloride in aqueous solution (pH 5.0 to 5.4) and400 ml of n-butanol. After allowing to separate the two phases, theaqueous layer containing the dissolved zinc hyaluronate is filteredthrough a membrane filter (0.45/μ pore size), then zinc hyaluronate isprecipitated by adding 3 volumes of ethanol, filtered on a glass filter,washed with ethanol and dried in a nitrogen atmosphere under mildconditions to obtain 0.82 g of zinc hyaluronate.

When necessary, a 0.50 % by wt./vol. solution is prepared from the zinchyaluronate obtained which is then further purified as described inExample 4. The characteristics of sodium hyaluronate used as startingmaterial are as follows:

    ______________________________________                                        Viscosity [η] .sub.25° C..sup.c→0 %                                              16.5 dl/g                                                Protein Content      018% by wt./vol.                                         ______________________________________                                    

Zinc hyaluronate can be prepared as described above also from associatesformed from other quaternary ammonium salts. Quaternary salts useful forthis purpose are e.g.:

a) carbotetradecyloxymethyl-trimethylammonium chloride (see theHungarian patent specification No. 188,537) ,

b) hexadecylpyridinium chloride,

c) cetylpyridinium chloride,

d) trimethylammonium chloride and the like.

EXAMPLE 7

Preparation of cobalt (II) hyaluronate

The process described in Example 6 is followed, except that thehyaluronic acid quaternary ammonium associate is dissolved in a solventcouple consisting of a 2% by wt./vol. cobalt(II)chloride. 6H₂ O aqueoussolution and n-butanol. The bond lengths are as follows: Co--O: 197pm±1.0 and Co--C: 239 pm+1.5 pm.

EXAMPLE 8

Preparation of an aqueous solution containing 0.50% by wt./vol. of zinchyaluronate made isotonic by zinc chloride

About 50 ml of a zinc chloride solution of 0.110 mol/liter concentrationare added to 0.50 g of sodium hyaluronate in a 100 ml volumetric flaskand then allowed to swell overnight. Then, the sodium hyaluronate isdissolved by shaking and the flask is filled up to the mark with a zincchloride solution of 0.110 mol/liter concentration.

The osmotic pressure of the solution obtained is 0.1491 mol/liter asexpressed in equivalent sodium chloride concentration, the value of pHis 5.0. When necessary, the pH value is adjusted to 5.5 to 5.6 by adding2.00 ml of a sodium acetate solution of 0.150 mol/liter concentration.After adjusting the pH value, the osmotic pressure of the solution is0.1489 as expressed in equivalent sodium chloride concentration.

The zinc hyaluronate solution is prepared from the particularly puresodium hyaluronate described in Example 3 with twice distilled waterunder aseptic conditions, then the solution is filtered through amembrane filter (0.45/μ pore size).

The solution obtained can be used in injectable compositions, too.

EXAMPLE 9

Preparation of an aqueous solution containing 0.2% by wt./vol. of zinchyaluronate made isotonic by zinc chloride

For a final volume of 100 ml, 0.20 g of sodium hyaluronate is weighed inand dissolved in a zinc chloride solution of 0.120 mol/literconcentration.

The dissolution and preparation of the zinc chloride solution ofprecisely 0.120 mol/liter concentration are carried out according toExample 1 according to the sense by changing the amount of zincchloride).

The osmotic pressure of the solution is 0.154 mol/liter as expressed inequivalent sodium chloride concentration; the pH shows a value of 5.3 to5.4.

    ______________________________________                                        HA content          1.96 mg/ml                                                Viscosity           15.9 dl/g                                                 Protein concentration                                                                             0.015 mg/ml                                               Purity of the solution.sup.x                                                                      A.sub.660.sup.1 cm = 0.015                                ______________________________________                                         .sup.x Based on the absorbance measured at 660 nm in a 1 cm cuvet.       

The solution is prepared by using the sodium hyaluronate of the qualitycharacterized in Example 2 and used first of all for the preparation ofdermatologic and cosmetic compositions.

EXAMPLE 10

Preparation of an aqueous solution containing 0.50 % by wt./vol. of zinchyaluronate made isotonic by glucose

The solution of this Example contains sodium hyaluronate and thecalculated equivalent amount of zinc chloride.

12.50 ml of a zinc chloride solution of 0.100 mol/liter concentrationare added to 0.50 g of sodium hyaluronate weighed in a 100 ml volumetricflask. (Another concentration of zinc chloride may also be used but theamount of zinc chloride should be the same.) Sodium hyaluronate isallowed to swell for 12 hours in the solution of zinc chloride filled upto 50 ml with ion-free water, then dissolved by shaking. Thereafter,24.50 ml of a glucose solution of 1.00 mol/liter concentration are addedand filled up to the mark with ion-free water.

The osmotic pressure of the solution is 0.1495 mol/liter as expressed inequivalent sodium chloride concentration; the pH shows a value of 5.4.Total zinc concentration=1.25×10⁻² mol/liter.

The solution is prepared by using the sodium hyaluronate of the qualitycharacterized in Example 2 and used first of all for the preparation ofdermatological and cosmetic compositions.

EXAMPLE 11

Preparation of an aqueous solution containing 0.2% by wt./vol. of zinchyaluronate made isotonic by glucose

The solution of this example contains sodium hyaluronate and thecalculated equivalent amount of zinc chloride.

5.0 ml of a zinc chloride solution of 0.100 mol/liter concentration areadded to 0.20 g of sodium hyaluronate weighted in a 100 ml volumetricflask, then the volume is completed to 50 ml with deionized water. Afterallowing to swell overnight, sodium hyaluronate is dissolved by shaking,27.0 ml of a glucose solution of 1.00 mol/liter concentration are addedand the flask filled up to the mark with ion-free water.

The osmotic pressure of the solution is 0.151 mol/liter as expressed inequivalent sodium chloride concentrations; the pH shows a value of 5.6to 5.7; Total zinc concentration=5×10⁻³ mol/liter.

EXAMPLE 12

Preparation of an aqueous solution containing 0.5% by wt./vol. of zinchyaluronate made isotonic by sorbitol

The zinc hyaluronate solution described hereinafter is prepared underaseptic conditions from sodium hyaluronate of particularly high puritydescribed in Example 3 and distilled water. The solution contains zincchloride in an equivalent amount calculated for sodium hyaluronate.

The process described in Example 10 is followed, except that, instead ofthe glucose solution, 23.50 ml of a sorbitol solution of 1.00 mol/literconcentration (182.19 g of D-sorbitol in 1 liter) are added to the zinchyaluronate solution.

The solution thus prepared is filtered through a membrane filter (0.45/μpore size). This solution can be used for any purpose includinginjectable compositions.

The osmotic pressure of the solution is 0.1520 mol/liter as expressed inequivalent sodium chloride concentration; the pH shows a value of 5.5;Total zinc concentration=1.25×10⁻² mol/liter.

EXAMPLE 13

Preparation of an aqueous solution containing 0.2% by wt./vol. of zinchyaluronate made isotonic by sorbitol

The solution described in this Example contains zinc chloride in anequivalent amount calculated for sodium hyaluronate.

The zinc hyaluronate solution described hereinafter is prepared underaseptic conditions from sodium hyaluronate of particularly high puritydescribed in Example 3 with twice distilled water.

The process of Example 12 is followed, except that 0.2 g of sodiumhyaluronate is dissolved, 5 ml of zinc chloride solution of 0.100mol/liter concentration, then 26.50 ml of a sorbitol solution of 1mol/liter concentration are added and finally, the solution is filled upto 100 ml. The solution thus prepared is filtered through a membranefilter (0.45/μ pore size) This solution can be used for any purposeincluding injectable compositions.

The osmotic pressure of the solution is 0.1501 mol/liter as expressed inequivalent sodium chloride concentration; the pH shows a value of 5.6.

    ______________________________________                                        Total zone concentration =                                                                         5 × 10.sup.-3 mol/liter                            Hyaluronate content  2.03 mg/ml                                               Viscosity            16.1 dl/g                                                Protein content      0.016 mg/ml                                              Purity of the solution.sup.x                                                                       A.sub.660.sup.1 cm = 0.010                               ______________________________________                                         .sup.x Based on the absorbance measured at 660 nm in a 1 cm cuvet.       

EXAMPLES 14 to 26

In the following the components of various compositions (pharmaceuticaland cosmetic compositions) are given in relation to formulation typesselected by us. The preparation of zinc hyaluronate solutions madeisotonic are described in the preceding Examples. Here, "distilled waterfor injection purposes" means twice distilled water prepared underaseptic conditions.

I. INJECTABLE SOLUTIONS

Compositions of Examples 14 to 17 are used for intracutaneousadministration whereas that of Example 18 serves for intraocular use.The active ingredient of the quality described in Example 3 is employedin these Examples.

EXAMPLE 14

    ______________________________________                                        Zinc hyaluronate active ingredient                                                                     2.0 mg                                               Sorbitol                 48.3 mg                                              Final volume of the aqueous solution                                                                   1.0 ml                                               prepared with distilled water for                                             injection purposes                                                            ______________________________________                                    

EXAMPLE 15

    ______________________________________                                        Zinc hyaluronate active ingredient                                                                     5.0    mg                                            Sorbitol                 42.8   mg                                            Final volume of the aqueous solution                                                                   1.0    ml                                            prepared with distilled water for                                             injection purposes                                                            ______________________________________                                    

EXAMPLE 16

    ______________________________________                                        Zinc hyaluronate active ingredient                                                                     2.0    mg                                            Propyl p-hydroxybenzoate 0.05   mg                                            Methyl p-hydroxybenzoate 0.5    mg                                            Glucose                  48.6   mg                                            Final volume of the aqueous solution                                                                   1.0    ml                                            prepared with distilled water for                                             injection purposes                                                            ______________________________________                                    

EXAMPLE 17

    ______________________________________                                        Zinc hyaluronate active ingredient                                                                     5.0    mg                                            Propyl p-hydroxybenzoate 0.05   mg                                            Methyl p-hydroxybenzoate 0.5    mg                                            Glucose                  44.1   mg                                            Final volume of the aqueous solution                                                                   1.8    ml                                            prepared with distilled water for                                             injection purposes                                                            ______________________________________                                    

EXAMPLE 18

    ______________________________________                                        Zinc hyaluronate active ingredient                                                                     10.0   mg                                            Potassium sorbate        1.0    mg                                            Sorbitol                 41.0   mg                                            Final volume of the aqueous solution                                                                   1.0    ml                                            prepared with distilled water for                                             injection purposes                                                            ______________________________________                                    

Compositions described in Examples 20 to 28 are mainly used fordermatologic and cosmetic purposes. The active ingredient of the qualitydescribed in Example 2 is employed in these Examples.

II. SOLUTIONS FOR TOPICAL USE EXAMPLE 19

    ______________________________________                                        Zinc hyaluronate active ingredient                                                                     5.0    mg                                            Potassium sorbate        1.0    mg                                            Sodium acetate           24.6   mg                                            Final volume of the aqueous solution                                                                   1.0    ml                                            prepared with distilled water                                                 ______________________________________                                    

EXAMPLE 20

    ______________________________________                                        Zinc hyaluronate active ingredient                                                                     2.0    mg                                            Potassium sorbate        1.0    mg                                            Sorbitol                 48.3   mg                                            Final volume of the aqueous solution                                                                   1.0    ml                                            prepared with distilled water                                                 ______________________________________                                    

III. GELS FOR TOPICAL USE EXAMPLE 21

    ______________________________________                                        Zinc hyaluronate active ingredient                                                                      20.0   mg                                           Acrylic acid polymerisate 200    mg                                           Sodium hydroxide of 30% concentration                                                                   50     mg                                           Potassium sorbate         10     mg                                           Distilled water   up to   10.0   mg                                           ______________________________________                                    

EXAMPLE 22

    ______________________________________                                        Zinc hyaluronate active ingredient                                                                      20.0   mg                                           Acrylic acid polymerisate 50     mg                                           Sodium hydroxide of 30% concentration                                                                   40     mg                                           Propylene glycol          1500   mg                                           Potassium sorbate         10     mg                                           Distilled water   up to   10.0   mg                                           ______________________________________                                    

IV. CREAMS AND OINTMENTS FOR TOPICAL USE EXAMPLE 23

    ______________________________________                                        Zinc hyaluronate active ingredient                                                                     50     mg                                            Potassium sorbate        10     mg                                            Soft white bee wax       125    mg                                            Sorbitan oleate          150    mg                                            Cetyl stearyl alcohol    840    mg                                            Glyceryl monostearate    1100   mg                                            Propylene glycol         4750   mg                                            Distilled water   up to  10     mg                                            ______________________________________                                    

EXAMPLE 24

    ______________________________________                                        Cobalt hyaluronate active ingredient                                                                   50     mg                                            Potassium sorbate        10     mg                                            Soft white bee wax       125    mg                                            Sorbitan oleate          150    mg                                            Cetyl stearyl alcohol    840    mg                                            Glyceryl monostearate    1100   mg                                            Propylene glycol         4750   mg                                            Distilled water   up to  10     mg                                            ______________________________________                                    

EXAMPLE 25

    ______________________________________                                        Zinc hyaluronate active ingredient                                                                     50     mg                                            2-Phenoxyethanol         100    mg                                            Sodium lauryl sulfate    100    mg                                            Cetyl palmitate          400    mg                                            Stearin                  400    mg                                            Stearyl alcohol          450    mg                                            Cetyl alcohol            450    mg                                            White vaseline           500    mg                                            Propylene glycol         550    mg                                            Glycerol                 600    mg                                            Distilled water   up to  10.0   mg                                            ______________________________________                                    

EXAMPLE 26

    ______________________________________                                        Cobalt(II)hyaluronate active ingredient                                                                 50     mg                                           2-Phenoxyethanol          100    mg                                           Sodium lauryl sulfate     100    mg                                           Cetyl palmitate           400    mg                                           Stearin                   400    mg                                           Stearyl alcohol           450    mg                                           Cetyl alcohol             450    mg                                           White vaseline            500    mg                                           Propylene glycol          550    mg                                           Glycerol                  600    mg                                           Distilled water   up to   10.0   mg                                           ______________________________________                                    

EXAMPLE 27

    ______________________________________                                        Zinc hyaluronate active ingredient                                                                     50.0   mg                                            Microcrystalline wax     250    mg                                            Propylene glycol         500    mg                                            Sorbitol                 400    mg                                            Wool wax (acetylated)    500    mg                                            White vaseline   up to   10     g                                             ______________________________________                                    

V. COMPOSITIONS FOR THE PURIFICATION AND CICATRIZATION OF PURULENTWOUNDS AND BURNS EXAMPLE 28

    ______________________________________                                        Zinc hyaluronate active ingredient                                                                     10     mg                                            Potassium sorbate        1.0    mg                                            Hydrophilic colloidal silicon dioxide                                                                  50     mg                                            Sorbitol   up to         1      mg                                            ______________________________________                                    

What is claimed is:
 1. A stoichiometric cobalt(II) hyaluronic acidcomplex prepared through the interaction of equivalent amounts of analkali metal hyaluronate, alkaline earth metal hyaluronate, silverhyaluronate or quaternary ammonium hyaluronate, and Co(II) ions inaqueous solution wherein the cobalt(II) is surrounded by four oxygenatoms, in the first coordination sphere, the length of the Co--O bonddistance is 197 pm±1.0 pm whereas two carbon atoms are present in alonger distance of 239 pm+1.5 pm from the cobalt atom.
 2. Apharmaceutical or cosmetic composition for local administration, whichcomprises as active ingredient, a pharmaceutically or cosmeticallyeffective amount of a stoichiometric cobalt(II) hyaluronic acid complexprepared through the interaction of equivalent amounts of an alkalimetal hyaluronate, alkaline earth metal hyaluronate, silver hyaluronateor quaternary ammonium hyaluronate, and Co(II) ions in aqueous solutionwherein the cobalt(II) is surrounded by four oxygen atoms, in the firstcoordination sphere, the length of the Co--O bond distance is 197 pm±1.0pm whereas two carbon atoms are present in a longer distance of 239pm+1.5 pm from the cobalt atom and a pharmaceutically acceptable inertcarrier.
 3. The stoichiometric cobalt(II) hyaluronic acid complexdefined in claim 1 prepared through the interaction of equivalentamounts of sodium hyaluronate and Co(II) ions in aqueous solution.